Roles Of ZNF84 In The Development And Progression Of HPV16-Related Cervical Squamous Cell Carcinoma

BackgroundThere is an etiologic relationship between high risk-human papillomaviruses (HR-HPVs) and cervical carcinoma. The sustained expression of E6, which is one of its early genes, is an important carcinogenic factor of HR-HPVs. The zinc finger protein family (ZNF) is closely related to the occurrence and evolution of a variety of tumors. ZNF84 is one of the important members of ZNF family. We found that the expression of ZNF84 was down-regulated after silencing E6 expression in HPV16 E6 positive cervical carcinoma cell line by Agilent human gene expression microarray. The relationship between HPV16 E6 and ZNF84 is still not clear.ObjectivesThe HPV16 E6-positive cervical squamous cell carcinoma tissues, cervical intra-epithelial neoplasia(CIN) tissues, cervical squamous epithelium tissues, cervical carcinoma cell line(cervical carcinoma SiHa cells), and the HPV16 E6-negative cervical squamous cell carcinoma tissues, CIN tissues, cervical squamous epithelium tissues, cervical carcinoma cell line(cervical carcinoma C33-A cells) were selected as the research subjects. The aims of this study are to investigate the expression and role of ZNF84 in the occurrence and evolution of HPV16 related cervical squamous cell carcinoma, and provide scientific evidence for further study on the prophylaxis and treatment of HPV 16 related cervical carcinoma.Methods1. The expression of ZNF84 was detected in HPV 16 E6-positive cervical carcinoma SiHa cells and HPV16 E6-negative cervical carcinoma C33-A cells by Western blotting assay.2. The immunohistochemical(IHC) staining method was used to tested the expression of ZNF84 in HPV16 E6 positive/negative cervical squamous cell carcinoma tissues, CIN tissues, cervical squamous epithelium tissues. The relationship between the expressions of HPV16 E6 and ZNF84 was analyzed also.3. The short hairpin RNA(shRNA) sequences targeting ZNF84 mRNA were designed by bioinformatics method. Then, the pGenesil-l-shRNA-ZNF84 recombinant plasmid and pGenesil-1-shRNA-vect control plasmid containing irrelevant sequence were constructed.4. The pGenesil-l-shRNA-ZNF84 and pGenesil-1-shRNA-vect plasmid were transfected into HPV16 E6-positive cervical carcinoma SiHa cells by liposomes. The SiHa cells transfected pGenesil-l-shRNA-ZNF84 plasmid were named as SiHa-shRNA-ZNF84 cells, while the SiHa cells transfected pGenesil-1-shRNA-vect plasmid were named as SiHa-shRNA-vect cells. The changes of migrative and invasive ability of HPV16 E6 positive cervical carcinoma SiHa cells before and after silenced the expression of ZNF84 were observed by transwell migration test and invasion test.Results1. The results of Western blotting results indicated that:HPV16 E6 was not expressed in cervical carcinoma C33-A cells, while HPV16 E6 was expressed in cervical carcinoma SiHa cells. ZNF84 was expressed in HPV16 E6 negative C33-A cells and HPV16 E6 positive SiHa cells, but the expression of ZNF84 in C33-A cells was significantly lower than that in SiHa cells(P<0.01)。2. The results from IHC revealed that:the expression of ZNF84 in HPV16 E6 positive cervical squamous epithelial tissues, CIN tissues and cervical squamous cell carcinoma tissues was significantly higher than that in HPV16 E6 negative cervical squamous epithelial tissues, CIN tissues and cervical squamous cell carcinoma tissues, and the expression of HPV16 E6 was positively correlated with ZNF84 (P<0.01).3. Silencing effect of shRNA targeting ZNF84 mRNA on ZNF84 expression in cervical carcinoma SiHa cells.The results of Western blotting displayed that:the expression of ZNF84 in SiHa-shRNA-ZNF84 cells was significantly lower than that in SiHa cells and SiHa-shRNA-vect cells (P<0.01), and the silence efficiency was 95.630%. Compared with SiHa cells, the expression of ZNF84 in SiHa-shRNA-vect cells was not significant difference (P>0.05).4. Effects of silencing ZNF84 expression on apoptotic and invasive ability of HPV16 E6 positive cervical carcinoma SiHa cells. The results from transwell migration test and invasion test illustrated that the number of SiHa-shRNA-ZNF84 cells through basement membrane of chamber was strikingly diminished than that of SiHa-shRNA-vect cells(P<0.01)Conclusions1. It’s proved that the expressions of ZNF84 and E6 in HPV16 E6 positive and negative cervical carcinoma cell line was positively correlated, as in HPV16 E6 positive and negative cervical tissue at different stages of the lesion.2. The ZNF84 silence could inhibit the migrative and invasive ability of HPV16 E6 positive cervical carcinoma SiHa cells.